A variety of inherited and acquired diseases are associated with genetic variations such as point mutations, deletions and insertions. Genetic variations such as single nucleotide polymorphisms may be informative in predicting response to drugs and providing prognostic indications of disease risk and severity. Some genetic variants are directly associated with the presence of disease, while others correlate with disease risk and/or prognosis. There are more than 500 human genetic diseases which result from mutations in single genes. These include cystic fibrosis, muscular dystrophy, α1-antitrypsin deficiency, phenylketonuria, sickle cell anemia or trait, and various other haemoglobinopathies. Furthermore, individuals with increased susceptibility to several common polygenic conditions, such as atherosclerotic heart disease, have been shown to have an association with the inheritance of a particular DNA sequence polymorphism. Cancer is thought to develop due the accumulation of genetic lesions in genes involved in cellular proliferation or differentiation.
The genetic variability within pathogens can play a role in the severity of associated disease and the nature of therapeutic intervention. Examples include (i) mutations associated with drug resistant strains of bacteria such as Tuberculosis; (ii) therapy induced resistance in viruses such as HIV which is associated with specific nucleotides, and (iii) specific sequences in HCV that are predictive of therapeutic response.
Genetic analysis is becoming routine in the clinic for assessing disease risk, diagnosis of disease, predicting a patient's prognosis or response to therapy, and for monitoring a patient's progress. The introduction of such genetic tests depends on the development of simple, inexpensive, and rapid assays for discriminating genetic variations.
Methods of in vitro nucleic acid amplification have wide-spread applications in genetics and disease diagnosis. Such methods include polymerase chain reaction (PCR), strand displacement amplification (SDA), helicase dependent amplification (HDA), Recombinase Polymerase Amplification (RPA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), transcription-mediated amplification (TMA), self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), or reverse transcription polymerase chain reaction (RT-PCR). Each of these target amplification strategies requires the use of oligonucleotide primers. The process of amplification results in the exponential amplification of amplicons which incorporate the oligonucleotide primers at their 5′ termini and which contain newly synthesized copies of the sequences located between the primers.
Commonly used methods for detection of small genetic variations involving PCR include High Resolution Melt curve analysis and the use of Molecular Beacons. Melt curve and Molecular Beacons are suitable methods for detection of sequences that represent a large proportion of the population, but they are not suitable for situations where the mutation must be detected in a large background of non-mutated DNA such as for acquired mutations involved in cancer, genotyping rare/emerging viral strains or identification of drug resistant bacteria in a background of drug sensitive bacteria.
As an example, PCR is extremely versatile and many modifications of the basic protocols have been developed. Primers used in PCR may be a perfectly matched to the target sequence or they can contain mismatched and or modified bases. Additional tag sequences at the 5′ end of primers can facilitate capture of PCR amplicons and the inclusion of labelled primers can facilitate detection. Other protocols which have introduced non-target related sequence (non-complementary tag sequence) into the 5′ portion of oligonucleotide primers have done so to introduce restriction sites for cloning or to tag amplicons for second round of amplification with generic primer which are not related to the original target. While it is known in the art that the 5′ half of a given primer can tolerate the insertion of bases that do not hybridize to the initial target, it also well acknowledged that the 3′ portion of the primer is far less amenable to the presence of mismatched bases.
This observation led to the development of oligonucleotide primers for Amplification Refractory Mutation System (ARMS) (Newton et al 1989 Nucleic Acids Research 17:2503-2516) also known as Allele Specific PCR (AS-PCR). ARMS primers promote discrimination of small genetic variations such as a single nucleotide polymorphism (SNP). This ability is based on the fact that oligonucleotides with a mismatched 3′ residue will not function as efficiently as primers compared to fully matched sequences. Kwok et al. demonstrated that this discrimination was not complete and depended on the DNA bases involved in the mismatch (Kwok, et al. 1990 Nucleic Acids Research 18: 999-10005). Double mismatches between a primer and template, with one mismatch at the 3′ end of the primer, provide an increased ability of ARMS primers to effectively discriminate between alleles (Kwok, et al. 1990 Nucleic Acids Research 18: 999-10005). ARMS primers must be well designed with the strength of the 3′ mismatch balanced by the strength of the second mismatch. This is also balanced by carefully selecting the annealing temperature of the PCR which has an effect on the efficiency with which mismatched primers anneal to their target. Design of ARMS assays can be difficult and development of reaction conditions, for example temperature, where all primers discriminate effectively is tedious.
Universal bases exhibit the ability to replace the four normal bases without significantly destabilizing neighboring base-pair interactions or disrupting the expected functional biochemical utility of the modified oligonucleotide. It is well known in the art that oligonucleotides that include a universal base will function as a primer for DNA sequencing or PCR. The most commonly used degenerate modified base is deoxyinosine, which serves as a “universal” base, as it is capable of wobble base pairing with all four natural nucleotides, though not with equal affinity (I-C>I-A>I-T˜I-G>I-I). As such, inosine has been extensively used in PCR in applications such as amplification of ambiguous sequences which require degeneracy at certain base positions of primers and probes.
WO 2006/092941 describes the use of dual specificity oligonucleotides composed of three different Tm portions to enhance specific amplification from PCR. The dual specificity oligonucleotide (DSO) is composed of 3 regions of sequence. The 5′ portion of the DSO is target-specific and has a high Tm. The middle portion of the DSO is a separation portion composed between 2 to 10 “universal” bases which are not any of the standard DNA bases (i.e. not G, A, T or C). The 3′ portion of the DSO is target-specific. DSO can tolerate mismatches within the 5′ and 3′ portions of the oligonucleotides and thus to use DSO as primers to effectively discriminate small genetic variations requires stringent conditions for primer annealing. The universal bases in the middle portion are capable of non-specific base paring with all four conventional DNA bases. As such these DSO primers are capable of binding to the initial target along the entire length of the primer (since the bases are universal as opposed to mismatched), although the temperature may be set such that the universal portion may not be bound during primer annealing. Once copied however, amplicons will contain variant sequence opposite the position of the universal bases and thus the presence of universal bases does not introduce any specific unique sequence into the amplicons generated using DSO primers.
Despite the relatively large number of techniques that have been developed for amplifying, detecting and analysing sequences, there is a substantial need for more rapid, accurate and inexpensive assays for discriminating genetic variations. A need also exists for methods that improve the amplification of sequences that comprise regions of genetic variability in cases where the variability is non-informative and complicates amplification. Further, better methods are required to increase the capacity to analyse more than one target in a multiplex format, particularly when detecting small genetic variations (e.g. single nucleotide polymorphisms—SNPs).